reperfusion injury in diabetic mice via modulation of gut microbiota

Author’s Accepted Manuscript Clostridium butyricum attenuates cerebral ischemia/reperfusion injury in diabetic mice via modulation of gut microbiota Jing Sun, Fangyan Wang, Zongxin Ling, Xichong Yu, Wenqian Chen, Haixiao Li, Jiangtao Jin, Mengqi Pang, Huiqing Zhang, Junjie Yu, Jiaming Liu

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S0006-8993(16)30185-8 http://dx.doi.org/10.1016/j.brainres.2016.03.042 BRES44808

To appear in: Brain Research Received date: 5 January 2016 Revised date: 29 February 2016 Accepted date: 28 March 2016 Cite this article as: Jing Sun, Fangyan Wang, Zongxin Ling, Xichong Yu, Wenqian Chen, Haixiao Li, Jiangtao Jin, Mengqi Pang, Huiqing Zhang, Junjie Yu and Jiaming Liu, Clostridium butyricum attenuates cerebral ischemia/reperfusion injury in diabetic mice via modulation of gut microbiota, Brain Research, http://dx.doi.org/10.1016/j.brainres.2016.03.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Clostridium butyricum attenuates cerebral ischemia/reperfusion injury in diabetic mice via modulation of gut microbiota Jing Sun a1, Fangyan Wangb1, Zongxin Lingc1, Xichong Yud1, Wenqian Chene, Haixiao Lie, Jiangtao Jine, Mengqi Pange, Huiqing Zhange, Junjie YUe, Jiaming Liu e* a Department of Neurology, the Second Affiliated Hospital of Wenzhou Medical University, 109 College West Road, Wenzhou, Zhejiang, 325027, China P.R. b Departments of Pathophysiology, Wenzhou Medical University, 1210 University Town, Wenzhou, Zhejiang, 325035, China P.R. c Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310003, China P.R. d School of Pharmaceutical Sciences, Wenzhou Medical University, 1210 University Town, Wenzhou, Zhejiang, 325035, China P.R. e School of Environmental Science and Public Health, Wenzhou Medical University, 1210 University Town, Wenzhou, Zhejiang, 325035, China P.R. * Corresponding author: Jiaming Liu, School of Environmental Science and Public Health, Wenzhou Medical University, 1210 University Town, Wenzhou, Zhejiang, 325035, China P.R. E-mail: [email protected]. Tel.: +86-577-8668-9848; Fax: +86-577-8669-9122. Abstract Diabetes is known to exacerbate cerebral ischemia/reperfusion (I/R) injury. Here, we investigated the effects of Clostridium butyricum on cerebral I/R injury in the diabetic mice subjected to 30 min of bilateral common carotid arteries occlusion (BCCAO). The cognitive impairment, the blood glucose level, neuronal injury, apoptosis, and expressions of Akt, phospho-Akt (p-Akt), and caspase-3 level were assessed. Meanwhile, the changes of gut microbiota in composition and diversity in the colonic feces were evaluated. Our results showed that diabetic mice subjected to BCCAO exhibited worsened cognitive impairment, cell damage and apoptosis. These were all attenuated by C. butyricum. Moreover, C. butyricum reversed cerebral I/R induced decreases in p-Akt expression and increases in caspase-3 expression, leading to inhibiting neuronal apoptosis. C. butyricum partly restored 1

These authors equally contributed to this work. 1

cerebral I/R induced decreases of fecal microbiota diversity, changes of fecal microbiota composition. Together, these findings highlight the important role of bacteria in the bidirectional communication of the gut–brain axis and suggest that certain probiotics might prove to be useful therapeutic adjuncts in cerebral I/R injury with diabetes. Keywords: diabetes; cerebral ischemia/reperfusion; Clostridium butyricum; apoptosis; gut microbiota; butyrate

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1. Introduction Cerebral ischemia/reperfusion (I/R) injury often causes neuronal injury and death, and results in functional impairment (Cumbler, 2015; Storm et al., 1985). Diabetes is a metabolic disease characterized by chronic hyperglycaemia, and high blood glucose level is one of the leading causes of ischemic stroke (Asfandiyarova et al., 2006; Chugunova et al., 2006). Hyperglycemia is the most important factor in the progression of diabetic stroke. The hyperglycemia can increase vulnerability to pathological changes, functional and biochemical alterations in the brain (Furie and Inzucchi, 2008; Heckmann et al., 2005). Epidemiologic studies have shown that diabetes is an important risk factor for strokes (Asfandiyarova et al., 2006; Hill, 2014; Shou et al., 2015). It was reported that patients with diabetes not only have a higher mortality rate, but tend to have more severe disability and slower recovery from stroke when compared to non-diabetic subjects(Cruz-Herranz et al., 2015). Ischemic stroke reveals a complex pathophysiological course that involves a plenty of distinct molecular and cellular pathways. Neuronal apoptosis, as a critical event during cerebral ischemia, mostly locates in the ischemic penumbra, which is potentially remedial (Air and Kissela, 2007; Martinez-Sanchez et al., 2007). After cerebral I/R injury, apoptotic signaling cascades were launched. Current researches have revealed that diabetes could aggravate cerebral I/R injury in animal models (Mendzheritskii et al., 2011; Pamenter et al., 2013), in particular, hyperglycemia in diabetic state increases occurrence of apoptosis in the brain (Kumar et al., 2014; Yu et al., 2015). Taking into account the facts that cerebral I/R injury is associated with the mechanism, to explore the neuroprotective agents that may attenuate the cerebral I/R damage with diabetes is of crucial importance.

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There is increasing, but largely indirect, evidence pointing to an effect of commensal gut microbiota on the central nervous system (CNS), suggesting that an interaction between the intestinal microbiota, the gut, and CNS in what is recognized as the microbiome–gut–brain axis (Burcelin et al., 2011). Furthermore, recent evidence indicates that the absence and/or modification of the gut microflora resulted in specified comorbidity between functional gastrointestinal disorders and CNS disease, such as traumatic brain injury(Bansal et al., 2009; Hang et al., 2003), psychological stress(Savignac et al., 2015), and Parkinson’s disease(Dutkiewicz et al., 2015; Kim and Sung, 2015; Mulak and Bonaz, 2015). Regulating the flora balance in the gut is achieved by the intake of probiotics, which is believed to be effective in lifestyle-related diseases (Camilleri, 2006; Cani et al., 2008). In recent years, it has been reported that probiotics can confer a healthy benefit to the development and function of brain in the host (Mikelsaar and Zilmer, 2009; Savignac et al., 2015). There are numerous evidences demonstrating that Lactobacillus and Bifidobacterium could attenuate anxiety, prevent the chronic psychological stress, reduce apoptosis in several brain regions and improve the learning and memory in mice(Messaoudi et al., 2011a; Messaoudi et al., 2011b). Thus, it proposed that the new role of gut microbiota could be associated with brain functions as well as neurological diseases via the gut-brain axis. Clostridium butyricum, notably beneficial probiotics, resides in the gut lumen and plays a protective role against pathogenic bacteria and intestinal injury (Kanai et al., 2015; Shinnoh et al., 2013). Recent evidence showed that C. butyricum effectively could exert the therapeutic effects on some human and experimental animal diseases, including antitumor effects by inducing apoptosis (Chen et al., 2015; Shinnoh et al., 2013). In our previous study, we also demonstrated that Clostridium

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butyricum could exert neuroprotective effects against vascular dementia (VaD) in mice via metabolic butyrate (Liu et al., 2015b). Moreover, our previous study also found that pretreatment with Clostridium butyricum displayed neuroprotective effects against cerebral I/R injury in mice (Sun et al., 2016). However, it is currently unclear whether potential probiotics such as C. butyricum could affect brain function, especially in cerebral I/R with diabetic animal. We hypothesized that gut-derived bacteria could play a role in cerebral I/R with diabetes. Thus, this present study was to assess the neuroprotection of C. butyricum on the cerebral I/R injury in diabetic mice, and investigated whether it exerted the neuroprotection by inhibiting neuronal apoptosis and modulating gut microbiota.

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2. Results 2.1 C. butyricum treatment decreased blood glucose level Before the behavioral test, the blood glucose level was measured. There were no significant differences of blood glucose level (P > 0.05, Fig. 1) between D-sham and D-I/R group. After the treatment with C. butyricum, the blood glucose level was significantly decreased compared with D-I/R group (P < 0.01, Fig. 1). 2.2 C. butyricum treatment ameliorated cognitive impairment The Morris water maze is conventionally used to measure cognitive function. In the training trials, all of the groups gradually learned to find the hidden platform and gently shortened their escape latency. A two-way ANOVA analysis showed that the escape latency in D-I/R group were significantly longer than in the D-sham group on days 2 (P < 0.05, Fig. 2A), 3(P < 0.05, Fig. 2A), 4(P < 0.01, Fig. 2A) and 5 (P < 0.01, Fig. 2A), suggesting an impairment in spatial learning of the D-I/R mice. After treatment with C. butyricum, the escape latencies on day 4 and 5 were significantly decreased compared with the D-I/R mice (P < 0.05, Fig. 2A).In the space exploration test, the data showed that there was obvious reduction of the time spent in the target quadrant in D-I/R group compared to the D-sham group (P < 0.01, Fig. 2B). The time spent in the target quadrant in the C. butyricum treatment group was much longer than that in D-I/R group (P < 0.05, Fig. 2B), indicating that C. butyricum treatment could attenuate cognitive impairment of D-I/R mice. 2.3 C. butyricum treatment ameliorated the histopathologic changes in the hippocampus H&E and TUNEL staining were used to investigate the neuronal injury and apoptosis in the hippocampus of mice. H&E staining revealed neuronal injury in ischemic penumbra of

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hippocampal CA1 region (Fig. 3). In the D-I/R group, hippocampal neuron exhibited shrunken neurons nuclear, disappearance of nucleolus, and disorder of the array of neurons (Fig. 3). After C. butyricum treatment, the histopathologic changes of the hippocampal neurons of D-I/R mice could be attenuated (Fig. 3). We analyzed neuronal apoptosis by TUNEL staining. The TUNEL positive cells were increased in the ischemic hippocampal region (Fig. 4 E). While in the C. butyricum treated groups had reduced TUNEL-positive staining than the D-I/R group (Fig. 4 E), suggesting decreased apoptosis by C. butyricum treatment. 2.4 C. butyricum treatment activated Akt Western blot analysis showed that cerebral I/R induced the sharp decreases in p-Akt expression levels in diabetic mice (P < 0.01, Fig. 4 A and B). Compared with D-I/R group, the expression of p-Akt was up-regulated after treatment with C. butyricum (P < 0.05, Fig. 4 A and B). The ratio of p-Akt/Akt in the C. butyricum-treated mice was significantly increased compared with the D-I/R group (Fig. 4 A and B), suggesting activating Akt by C. butyricum treatment. 2.5 C. butyricum treatment resulted in decreases of caspase-3 protein By western blot, cerebral I/R induced the sharp increases in caspase-3 expression level in diabetic mice. Compared with D-I/R group, the expressions of caspase-3 were down-regulated after treatment with C. butyricum (P < 0.01, Fig. 4 C and D). The protein level of caspase-3 in the D-I/R group was significantly increased compared with the D-sham group (P < 0.01, Fig. 4 C and D), whereas that of the C. butyricum-treated mice was significantly decreased compared with the D-I/R mice (P < 0.01, Fig. 4 C and D).

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2.6 Structural modulation of the fecal predominant microbiota In that alterations in the gut microbiota have been linked with behavior, cecal microbiota by PCR-DGGE was assessed. PCR-DGGE profiles showed the overall structure and diversity of the fecal predominant bacteria. The numbers of bands in D-I/R mice were fewer than that in the D-sham group, indicating a dramatically decreased predominant bacterial diversity. However, there were dramatic changes in the richness and diversity of the fecal predominant bacteria in the C. butyricum-treated group, which indicated the restoration of fecal microbiota after C. butyricum treatment (Fig. 5 A). The cluster analysis of the DGGE profiles, which was based on the similarity indices, showed that samples from the D-sham mice and the C. butyricum-treated mice clustered together in one branch, whereas the samples from the D-I/R mice clustered in a separate branch, indicating the restoration of fecal microbiota after C. butyricum treatment (Fig. 5 B). 2.7 Quantification of predominant fecal bacteria by qPCR. The specific modulations of the fecal microbiota, nine predominant bacteria were detected. The differences in predominant fecal bacteria after treatment with C. butyricum were detected by qPCR. Our data demonstrated that cerebral I/R induced the sharp decreases in Bacteroides/prevotella,

Clostridium

cluster

XIVab,

Fecalibacterium.

prausnitzii,

Bifidobacterium, and Lactobacillus in the D-I/R group and marked increases in Clostridium cluster XI, Clostridium cluster I, Enterobacteriaceae and Enterococcus spp in the D-I/R group (Fig. 6 (a) ~ (i)). Of interest were the findings that Clostridiumcluster XIVab, F. prausnitzii, Bifidobacterium, and Lactobacillus were obviously increased and Clostridium cluster XI, Clostridium cluster I, Enterobacteriaceae and Enterococcus spp were significantly

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decreased in the D-I/R mice by C. butyricum treatment (Fig. 6 (a) ~ (i)), suggesting a positive effect of C. butyricum on microbiota and brain function.

3. Discussion Probiotics are dietary supplements that exert beneficial effects under various clinical conditions including antimicrobial-associated diarrhea(Kim et al., 2003; Vanderhoof and Young, 1998), irritable bowel syndrome（IBS）(Camilleri, 2006)and dysbacteriosis-related diseases (diabetes)(Kasinska and Drzewoski, 2015; Stenman et al., 2015) in humans and animals. However, the widespread use of probiotics for cerebral I/R injury in diabetes is lacking, due to little mechanisms insight (Delzenne et al., 2015). This study may be the first time to demonstrate specifically and previously undescribed neuroprotective effects induced by modulation of intestinal microbiota using a potential probiotic [C. butyricum] in the D-I/R mice. As the results shown apparently, C. butyricum treatment significantly reversed the behavioral deficits in the Morris water maze test and decreased the high level of blood glucose in the D-I/R mice. Meanwhile, reducing cognitive dysfunctions and histopathologic changes in the model mice were also observed. And the molecule alterations of apoptotic related proteins (p-Akt and caspase-3) in the brain were reversed by the treatment with C. butyricum in the model mice. Furthermore, C. butyricum treatment could dramatically augment the descending microbiome diversity in the D-I/R mice. Therefore, the neuroprotective properties of C. butyricum treatment on the cerebral I/R injury in diabetes may be involved in the modulation of gut microbiota. To develop cerebral I/R injury in diabetic mouse model, the STZ-induced diabetic mice

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were subjected to 30 min of BCCAO, which can lead to damage of cognitive deficits and hippocampal neurons (Gillingwater et al., 2004; Wu et al., 2001). Consistent with our results, in the Morris water maze, the escape latency and the time spent in the target quadrant significantly prolonged in the D-I/R mice, suggesting that cognitive impairment occurred in the D-I/R mice in diabetes. However, C. butyricum treatment could shorten the escape latency and the time spent in the target quadrant in the D-I/R mice in diabetes, suggesting that C. butyricum could alleviate cognitive deficits in the D-I/R mice. At the same time, histopathologic changes in the brain were likely to be pallel to the alteration of behaviors. It is widely acknowledged that diabetes is a high-risk factor for ischemic stroke, and numerous studies have demonstrated that hyperglycemia exacerbates neuronal damage initiated by cerebral ischemia (Baliga and Weinberger, 2006; Furie and Inzucchi, 2008). The CA1 neurons in hippocampal are always sensitive to the fluctuation in the brain, such as ischemia, anoxia and so on. In line with our results, morphological changes, including disappearance of nucleolus and shrunken neurons due to condensation of cytoplasm and karyoplasms, in the hippocampal CA1 neurons were present extensively in the D-I/R mice. By the contrast, C. butyricum treatment significantly reversed neuronal changes, suggesting that C. butyricum treatment could ameliorate the histopathologic changes in the CA1 region of hippocampus. Hyperglycemia is the most important factor in the progression of diabetic stroke (Chugunova et al., 2006), and cerebral I/R injury in diabetes results in ultimately activating an apoptotic cascade, immediately after the onset of reperfusion (Liu et al., 2015a; Wang et al., 2001). Apoptosis can either be physiological or pathophysiological cell death and this form of cell death is defined by a distinct set of both biochemical and morphological characteristics

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including cell volume decrease, Caspase activation, chromatin condensation, DNA laddering, and cell fragmentation(El-Sahar et al., 2015; Park et al., 2015). In this study, we observed a reduction of apoptosis in C. butyricum-treated mice using TUNEL staining. These findings could partially account for the neuroprotective effects observed in C. butyricum-treated mice. To gain mechanistic insight, we investigated the expression patterns of PI3K/Akt pathway-related proteins in the brain. The PI3K/Akt signaling pathway, which is a central mediator to regulate neuronal growth, survival, and metabolism, has been widely reported to participate in the protection against psychiatric disorders (Qu et al., 2015; Wang et al., 2007). In line with our results, C. butyricum treatment could activate Akt and increase the p-Akt level, suggesting that C. butyricum can activate PI3K/Akt pathway. Akt diverges into multiple signaling cascades. And its effects on promoting cell survival by inhibiting apoptosis are versatile. Caspase-3, the major targets, plays a fundamental role in the development of both I/R injury and diabetes(Duan et al., 2015). It is well known that caspase-3, a key factor in apoptosis, can activate a DNA fragmentation factor which activates endonucleases to cleave nucleicacids and finally lead to cell death (Liu et al., 2008). A large of body evidences have indicated that the up-regulation of caspase-3 is found after cerebral ischemia (Li et al., 2015). In this study, ischemic brain injury induced an increase of caspase-3 level, while treatment with C. butyricum could significantly attenuated cerebral I/R injury-induced activation of caspase-3. Taken together, it suggested that C. butyricum treatment could attenuate apoptosis in the D-I/R mice in diabetes via decreasing the levels of caspase-3, in response to PI3K/Akt activation. The molecular mechanisms underlying how the bacteria affects neuronal apoptosis in the D-I/R mice in diabetes needs to be resolved in future studies.

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Accumulating evidence from animal studies supports the hypotheses that gut microbiota play an important role in central nervous system (CNS) function (Bercik et al., 2012; Savignac et al., 2014). Moreover, it is worth noting that psychiatric disorders, such as psychological stress (Savignac et al., 2015), and Parkinson’s disease(Dutkiewicz et al., 2015; Mulak and Bonaz, 2015) could lead to gastrointestinal dysfunction, even causing compositional changes in gut microbiota. Thus, it proposed that the new role of gut microbiota could be associated with brain functions as well as neurological diseases via the gut-brain axis (Mayer et al., 2015). It is worth noting that the majority of studies on the microbiome–gut–brain axis are focused on affecting gut microbiota under the psychological status (Luna and Foster, 2015). However, gut-brain axis is defined as two-ways regulative pattern between the gut and brain. Currently, increasing evidences have shown that probiotics could offer promise for a wide diversity of diseases and may be involved in the maintenance of a healthier composition and diversity of gut microbiota (Luna and Foster, 2015; Mulak and Bonaz, 2015; Savignac et al., 2015). Lactobacillus spp and Bifidobacterium spp have been reported to reverse cerebral I/R injury, attenuate anxiety and chronic psychological stress, reduce apoptosis in several brain regions and improve learning and memory in mice (Messaoudi et al., 2011b). Moreover, our previous study demonstrated that C. butyricum displayed neuroprotective effects against ischemic vascular dementia in non-diabetic mice by regulating gut microbiota. And probiotics have also been identified as effective adjuvants in insulin resistance therapies, which are known to reduce blood glucose levels and decrease the complications of diabetes (Hu et al., 2015; Kootte et al., 2012). In this study, we observed that C. butyricum-treated mice significantly increased bands in the DGGE profiles compared to

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D-I/R mice, suggesting that C. butyricum can improve the diversity of gut microbiota in D-I/R mice.

qPCR

results

showed

Bacteroides/prevotella,

Clostridium

cluster

XIVab,

Fecalibacterium. prausnitzii, Bifidobacterium, and Lactobacillus in the D-I/R group were significantly decreased compared with that in the D-sham group, while, after C. butyricum treatment, Clostridiumcluster XIVab, F. prausnitzii, Bifidobacterium, and Lactobacillus were significantly restored, which is even similar to the diversity of gut microbiota in D-sham group. In the present work, our data demonstrate that C. butyricum can exert neuroprotective effects relevant to partly restoring the microbiota composition in the D-I/R mice. Considerable further research needs to be examined to the molecular mechanisms at a microbiome level underlying the neuroprotective effects observed. Moreover, future studies using dead bacteria, killed in such a way as to exclude microbiome structural alteration, are needed to further insight into the mechanism of action of the probiotics. Interestingly, our results showed that there was a significant decrease of blood glucose level in the C. butyricum-treated group. Diabetes is a metabolic disease characterized by chronic hyperglycaemia and high blood glucose level is one of the leading causes of ischemic stroke (Air and Kissela, 2007; Asfandiyarova et al., 2006). So, an efficient blood glucose control strategy is crucial for effective ischemic stroke in diabetes (Chugunova et al., 2006). Meanwhile, diabetes is a condition of multifactor including intestinal microbiota interacts. An imbalance of the microbiota or an enrichment of specific bacterial strains in the gut has been associated with diabetes(Gomes et al., 2014). The effects of probiotics on various gut-related diseases have been investigated extensively in several in vitro and in vivo experiments and inhuman clinical trials(Delzenne et al., 2015). Recent studies have shown that Lactic acid

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bacteria, in particular, are known to reduce blood glucose levels and decrease the complications of diabetes (Honda et al., 2012; Yakovlieva et al., 2015; Yun et al., 2009). In the present study, treatment with C. butyricum did influence glucose level in the D-I/R mice. We would like to propose that one of the beneficial effects of C. butyricum to attenuate the cerebral I/R injury in diabetic is, at least in part, due to the lowering of blood glucose level. However, this hypothesis requires further investigations. So far, the molecular mechanisms underlying C. butyricum action on brain remain fully unclear. Changes occurring in the brain following gut microbiota modulation may come from a humoral, hormonal or neuronal route; and this, via through metabolic molecules directly traveling up to the brain (via the bloodstream), or signals transmitted up to the brain (Dinan et al., 2013; Rhee et al., 2009). Short chain fatty acid (SCFA) including principally acetate, propionate and butyrate, are considered to be neuroactive microbial metabolites that can cross the blood-brain barrier and modulate CNS functions (Macfabe, 2012). Indeed, probiotics can modify the gut flora and result in changes in the levels of SCFA butyrate (Yadav et al., 2013), which has been reported to improve spatial learning and memory ability(Yoo et al., 2015), alleviate ischemic stroke(Sun et al., 2015), and provide anti-apoptotic and anti-oxidant effects (Kim et al., 2009). Furthermore, GLP-1 secretion has been associated with gut flora modulation in other studies (Cani et al., 2009). The probiotic VSL#3 treatment resulted in modulation of gut flora composition and a rise in the levels of butyrate, which stimulated the release of GLP-1 from intestinal L-cells, resulting in improved glucose tolerance (Yadav et al., 2013). Moreover, gut microbiota modulation have been also correlate with changes in levels of various neurotrophins and monoamine neurotransmitters involved in brain function

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(Desbonnet et al., 2014). However, currently it is impossible to provide the overall mechanisms of C. butyricum neuroprotective effects, due to many molecules may be involved in. In conclusion, in present study, we demonstrated that administration of C. butyricum exerted beneficial effects against cognitive impairment, histopathological changes, and apoptosis in the D-I/R mice, and molecule mechanism of anti-apoptosis might be relate to activate PI3K/Akt pathway. In the PI3K/Akt pathway, the altered expression of apoptotic related proteins (Akt, p-Akt, and caspase-3) in the brain were reversed after the treatment with C. butyricum. Meanwhile, C. butyricum treatment also reversed the descent of microbiome diversity in the cerebral I/R injury with diabetes. And lowering of blood glucose level due to modulating gut microbiome might play a major role in attenuating the cerebral I/R injury in diabetic. Furthermore, our current studies possibly offer the intriguing opportunity of developing unique microbial-based strategies for the adjunctive treatment of cerebral ischemic injury in diabetes.

4. Methods and materials 4.1 Animals Male C57BL/6 mice (six weeks old, 20 to 22g) were purchased from the Experimental Animal Center of Wenzhou Medical University and maintained under specific pathogen–free (SPF) conditions. Mice were housed in groups (3 – 4) with ad libitum access to food and water under controlled laboratory conditions of temperature（22 ± 2 °C）and humidity (55 ± 5 %) with a 12 h light/dark cycle (lights on at 08:00 am). The mice were allowed to

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acclimatize to the laboratory for 1 week prior to the beginning of study. Animals were housed in a specific room, and the treatment groups were separated from each other to avoid cross contamination. All experiments were performed according to Animal Use guidelines and approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University. 4.2 Induction of diabetes and cerebral I/R model After fed with a high-fat diet for four weeks, mice were given streptozotocin (STZ, Sigma Chemical Co. St. Louis, USA. 50 mg/kg body weight) intraperitoneally. After 10 days of STZ injection, the blood glucose levels were measured and mice with glucose levels above 16.7 mmol/L were enrolled as diabetic. The diabetic mice were monitored for blood glucose levels for at least two weeks to ensure the stability of the animal model. Then, the diabetic mice model were subjected to 30 min of bilateral common carotid arteries occlusion (BCCAO) with vascular clips, leading to cerebral I/R injury, which is called cerebral I/R injury in diabetic mouse model. The procedure of BCCAO was performed as described previously (Espinera et al., 2013; Terashima et al., 1998), as following: firstly, the mice were anaesthetized with 400 mg/kg injection of chloral hydrate and a midline incision in the ventral side of the neck was made to expose the right and left common carotid arteries; secondly, we gently separated the two arteries and occluded them with vascular clips for 30 min, and then the clips were removed to restore blood for recirculation; after 2 h of occlusion, the filament was withdrawn to enable reperfusion. The sham-operated mice underwent the same operation procedure, but the common carotid arteries were not occluded. 4.3 Bacterial preparation C.butyricum WZMC1016 (CGMCC 9831) was provided by the China General

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Microbiological Culture Collection Center and was cultured in MRS broth (Hopebio, Qingdao, China) for 24 h in an anaerobic chamber (5 % CO2) at 37 °C. C. butyricum were harvested from the MRS broth (4, 500 rpm; 15 min) and resuspended in sterile saline. We established the standard curve between the absorbance and colony forming units (CFU) with a positive linear relationship, giving calculated bacteria counts of 5.0 × 109 CFU /mL at an absorbance of 0.25 at 600 nm. The final experimental concentration was 5.0 × 108 CFU/mL. 4.4 Experimental design The diabetic mice were randomly divided into three groups, n = 12 for each group: (1) diabetic sham (D-sham) group, which was subjected to a sham operation; (2) diabetic I/R model group (D-I/R), which was subjected to cerebral I/R injury; (3) C. butyricum-treated group (D-I/R +C. b), which were subjected to cerebral I/R injury and treated with a suspension of C. butyricum (1 × 108 CFU) that was freshly prepared as previously described. The D-sham control group and D-I/R model groups were treated with physiological saline. All animals were treated intragastrically with physiological saline or C. butyricum following BCCAO at doses of 200 μL through a stainless steel gavage needle once daily for 6 weeks (the initial treatment was 24 h after the BCCAO operation). At the fifth week, behavioral tests were performed by Morris water maze. During the behavioral evaluations, C. butyricum was administered after the tests every day. During the experiment, body weight and blood glucose level were monitored weekly. At the end of the experimental period, the mice were euthanized and tissue samples (brain tissue and colonic feces) were collected for analysis. 4.5 Morris water maze Evaluation of cognitive impairment was performed at the fifth week (n = 8 – 10). The Morris

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water maze (DigBehv-MG, Shanghai Jiliang Software Technology Co., Ltd., China) consisted of a video capture system, a data analysis system and a circular plastic water tank (Φ = 120 cm). The tank was filled with water at a depth of 30 cm, and divided into four equal hypothetical quadrants. The position of marks remained unchanged throughout the test. A platform (Φ = 9 cm) was placed into the water at a constant position and submerged 1 cm below the surface of the water. The Morris water maze experiment assesses positional navigation and spatial exploration. In training experiment, on days 1 – 4, mice were placed in the water at 4 distinct starting quadrant points and trained to find the submerged platform within 60 s. The time spent finding the platform is termed as the escape latency. On the 5th day, the platform was removed from the tank and each mouse was tested in a probe trial in which the time spent in the former platform-containing quadrant was recorded. The Morris water maze testing procedure was performed as previously described. 4.6 Histology Brain tissues were fixed, then dehydrated using ascending grades of ethyl alcohol, and cleaned in xylene and embedded in paraffin. Cross sections of about 5 μm thickness were cut with a microtome, mounted on glass slides and stained with: Hematoxylin and eosin (H&E) and TUNEL (TUNEL, Roche Diagnostics, Germany) according to the manufacturer’s instructions, as described previously (Sun et al., 2015). The TUNEL-positive cells were brown cells with apoptotic nuclear features. The sections were then examined under light microscope for histological changes. 4.7 Western blot Brain samples were rapidly dissected, rinsed in 1 × PBS, and homogenized in an ice-cold

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homogenization buffer (Beyotime Institute of Biotechnology, Shanghai, China). The samples were then analyzed by Western blot, as described previously(Sun et al., 2015). The proteins were collected and centrifuged at 12, 000×g for 15 min at 4 °C. A BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to determine the protein concentration. The samples (20 μg) were separated using 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were incubated in blocking solution comprising 5% non-fat milk in TBST at room temperature for 1.5 h and then incubated with the primary antibodies diluted in blocking solution individually. The primary antibodies were anti-Akt, anti-p-Akt, and anti-caspase-3 antibody (1: 1000, Bioworld, USA). After incubation with primary antibodies; membranes were incubated with HRP conjugated secondary antibodies (1: 2000, Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at room temperature. The bound antibodies were detected by a chemiluminescence system (ECL Plus, Thermo Scientific, and Rockford, IL). Images were scanned and the results were quantified using the National Institute of Health Image J software (Bio-Rad Laboratories, Hercules, CA, USA). β-actin was performed as a internal control. 4.8 PCR-DGGE Analysis The fecal samples were collected and frozen at -80°C. Bacterial DNA was extracted using a QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions (Sun et al., 2015). DGGE analyses of the PCR products were performed by using a D-Code system (Bio-Rad, Hercules, CA, USA).

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4.9 qPCR for fecal predominant bacteria The qPCR assay was performed with a Power SYBR Green PCR Master Mix (Takara, Dalian, China) on ABI ViiA7 real-time PCR system (Applied Biosystems, Carlsbad, CA) according to the manufacturer’s instructions. Bacterial specific primer sets and the reaction conditions used for qPCR were performed according to previous study. The copy number of target DNA was determined by comparison with a 10-log-fold diluting standards plasmid DNA running on the same plate. All reactions were carried out in triplicate repeats and a nontemplate control was performed in every analysis. Bacterial quantity was presented as log 10 bacteria per gram of feces (wet weight). 4.10 Statistical analysis All data are presented as the mean ± SEM and the differences between groups were evaluated by two-way ANOVA or one-way ANOVA by Tukey’s test. Statistical analyses were performed using SPSS statistical software, version 17.0 (SPSS, Chicago, IL, USA). P < 0.05 was considered significant. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments The research was supported by Wenzhou Science and Technology Development Funds (Y20150174).

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Figure legends Fig.1 Effect of C. butyricum on blood glucose level. Values are presented as the mean ± SEM. D-sham, diabetic sham-operated group; D-I/R, diabetic I/R model group; D-I/R+Cb, which were subjected to diabetic I/R and treated with C. butyricum. ##P < 0.01 vs. D-I/R group. Fig.2 Morris water maze test. (A) Escape latency (s). (B) The time spent in the target quadrant (s). Error bars indicate SEM. *P < 0.05 vs. D-sham group, **P < 0.01 vs. D-sham group and #

P < 0.05 vs. D-I/R group.

Fig.3 Effect of C. butyricum on histopathology. Representative photomicrographs of H&E staining. An arrow indicates injured neurons. Magnification: 400 ×. Scale bar = 20 µm. Fig.4 Effect of C. butyricum on the neuronal apoptosis. (A) Western blot studies of p-Akt; (B) the ratio of the protein levels of the p-Akt /Akt; the reference value of the p-Akt /Akt ratio was the ratio of the D-sham group; (C) Western blot analysis of caspase-3 expression, levels were normalized to levels of the internal control (β-actin); (D) quantitative analysis of expression of caspase-3, the reference value of the caspase-3/β-actin was the ratio of the D-sham group. Error bars indicate SEM; n = 6 for each group. *P < 0.05 vs. D-sham group and **P < 0.01 vs. D-sham group; #P < 0.05 vs. D-I/R group and ##P < 0.01 vs. D-I/R group. (E)

Representative

photomicrographs

of

TUNEL

staining.

An

arrow

indicates

TUNEL-positive neurons. Magnification: 400 ×. Scale bar = 20 µm. Fig.5 PCR-DGGE analysis of the predominant fecal microbiota in mice. (A) PCR-DGGE fingerprints used to analyze the fecal microbiota of the sample from the D-sham group (A1 ~ A5), D-I/R group (B1 ~ B5) and D-I/R + Cb group (C1 ~C5). Each lane represents one subject that was randomly selected from each group. The bands marked in the DGGE gel

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were identified by cloning and sequencing to facilitate the interpretation of the figure. (B) Dendrogram of the DGGE profiles. Fig.6 Quantification of predominant fecal bacteria by qPCR. (log10 copies/g fresh feces). The predominant fecal microbiota was detected. Error bars indicate SEM; n = 6 for each group. *P < 0.05 vs. D-sham group, **P < 0.01 vs. D-sham group, #P < 0.05 vs. D-I/R group and ##P < 0.01 vs. D-I/R group.

Highlights 

C. butyricum displayed neuroprotective effects in cerebral I/R in diabetic mice.



C. butyricum exerts protective effects via anti-apoptosis of Akt activation.

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C. butyricum restored cerebral I/R induced decreases of fecal microbiota diversity.



A microbial-based strategy is useful to cerebral I/R injury in diabetic mice.

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